2D and E). for ~80% of lung malignancies (1). Activating mutations of the epidermal growth factor receptor (EGFR) occur in 30C40% of the patients with NSCLC in China, and are associated Rabbit polyclonal to ACBD6 with poor prognosis (2). EGFR mutations result in constitutive activation of the EGFR in the absence of the EGF ligand and abnormal activation of downstream signaling pathways, including mitogen-activated protein kinase (Mek)/extracellular signal regulated kinase (ERK) and phosphatidylinositol-3 kinase (PI3K) (3C5). Activation of these downstream effectors upregulates Mcl-1, Bcl-XL and survivin, allowing malignancy cells to evade apoptosis (6C8). EGFR inhibitors have recently been used clinically to improve the poor prognosis of NSCLC with EGFR mutations. Almost 90% of these somatic activating mutations in EGFR consist of in-frame deletions in exon 19 and L858R point mutations in exon 21 (9,10). Gefitinib, a synthetic anilinoquinazoline, is an orally active and highly selective EGF receptor inhibitor that blocks EGF receptor autophosphorylation and subsequent signal transduction pathways implicated in the promotion of cancer cell proliferation (11). At present, gefitinib is applied to a number of human cancers and benefits some patients CP-91149 during treatment (12). However, while most NSCLC patients with EGFR CP-91149 mutations initially respond to EGFR-tyrosine kinase inhibitors (TKIs), acquired resistance ultimately develops (13). One potential explanation for the acquired resistance may be a secondary mutation in EGFR, EGFR T790M, which occurs in ~50% of patients acquiring resistance to EGFR-TKIs (14). Additionally, MET oncogene amplification occurs in 20% of the patients with EGFR-TKI resistance (15). Amplification of MET was found to cause gefitinib resistance by driving ERBB3 (HER3)-dependent activation of PI3K, a pathway thought to be specific CP-91149 to the EGFR/ERBB family receptors (16). Through CP-91149 genetic changes, malignancy cells acquire a survival advantage, such as resisting drug-induced apoptosis, to decrease the sensitivity to drug therapy. Nevertheless, this durable genetic resistance takes a relatively long time to develop, whereas other temporary or poor types of resistance mechanisms come into play earlier in treatment (17). There is evidence that this behavior of carcinomas is usually influenced by crosstalk between tumor cells and the host microenvironment (18,19). Stromal cells reduce the sensitivity of cancer cells to chemotherapy drugs (20,21), leading to the suggestion that co-culture of cancer cells with stromal cells may cause reduced gefitinib-induced apoptosis in cancer cells. Since fibroblasts play a definitive role in tumor progression and drug response (22,23), in the present study we confirmed that fibroblasts efficiently induced gefitinib resistance in the HCC827 cell line which expresses EGFR exon 19 deletion mutations. In order to investigate how the susceptibility of lung cancer cells with EGFR-activating mutations to an EGFR-TKI could be affected by fibroblasts, we performed bioinformatic analysis and found that Aurora-A kinase (AURKA) overexpression played an important role in the reduced apoptosis in HCC827 cells. Further investigations showed that this p53 pathway may play a key role in the regulation of gefitinib resistance. Materials and methods Cell lines and reagents We purchased EGFR-mutant human lung adenocarcinoma cell line HCC827 (del E746_A750) and human lung embryonic fibroblast MRC-5 cells from the Cell Bank of the Chinese Academy of CP-91149 Sciences. We maintained the cell lines in RPMI-1640 medium made up of 10% FBS (Gibco-BRL, Gaithersburg, MD, USA) at 37C in a humidified 5% CO2 atmosphere. To study the effect of cancer-associated fibroblasts on gefitinib sensitivity of HCC827 cells, one patient with histologically confirmed lung cancer and who underwent surgical resection in.